Entomological Exploration of Sand Flies in Human Communities Affected by Cutaneous and Visceral Leishmaniasis in El Hajeb Province, Morocco

Sand flies are the exclusive vectors of leishmaniasis. This group of parasitic diseases is a serious public health problem in Morocco. The aim of this study was to investigate the sand fly fauna, mainly the species composition, biodiversity, and seasonal activity of sand flies in El Hajeb in central Morocco. A total of six stations (Aït Naaman, Aït Rbaa, Aït Brahim, Ain Taoujdate, Sidi Mbarek, and Aït Oufella) were studied, five of which had recently recorded cases of leishmaniasis. Sand fly bimonthly captures were carried out using a sticky paper trap in different biotopes from March to December 2019. A total of 14590 adult sand flies were collected. The activity of the sand fly started in April and declined in November. The periods of high abundance were July, September, and November. Morphological identification of sand flies shows the presence of twelve species: Phlebotomus papatasi, P. longicuspis, P. perniciosus, P. sergenti, P. bergeroti, P. alexandri, P. dreyfussi, P. ariasi, Sergentomyia fallax, S. minuta, S. schwetzi, and S. antennata. The analysis showed that species belonging to the genus Phlebotomus were the most dominant (93.3%) and the risk periods were spread during the summer and autumn seasons. The present study provides for the first time information on the species of sand flies in El Hajeb. It, therefore, provides decision makers with an important tool to conduct vector control actions during peak periods in order to limit the transmission of leishmaniasis. A preprint was made available by the research square in the following link: “https://assets.researchsquare.com/files/rs-1409330/v1/dfef7013-0327-4a54-897f-214924a2d950.pdf?c=1646838874.”


Introduction
Sand fies (Diptera, Psychodidae, and Phlebotominae) are widely distributed in tropical and temperate climates and are of great medical and veterinary importance. Tey are currently considered as the exclusive vectors of human and canine leishmaniasis [1]. Tese diseases are ranked second only to malaria as vector-borne diseases threatening global health [2]. Tey represent a complex group of parasitic diseases, both clinically and ecoepidemiologically [1,2].
In Morocco, leishmaniasis is a serious health problem and is endemic in several regions of the country, manifesting itself in two main clinical forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) [3,4].
From the epidemiological point of view, the main foci of the visceral and cutaneous forms, which are caused by the Leishmania infantum (L. infantum) parasite, have been recorded mainly in the north of the country, while the outbreaks of the wet cutaneous forms, which are caused by Leishmania major (L. major), were reported in the south of the country; and those of the dry cutaneous forms, which are caused by Leishmania tropica (L. tropica), were reported mainly in the center of the country [3,5].
Beyond this clinical and parasitic variety of leishmaniasis, their common point is that they are transmitted in natural conditions by the bite of diferent species of infected female sand fies [1]. Tese insects are called "Chniwla" in most regions in Morocco [6][7][8].
From an entomological point of view, not only the vectorial capacity of sand fies of the genera Phlebotomus in the old world and Lutzomyia in the new world has been demonstrated for the transmission of leishmaniasis but also that of other viral or bacterial diseases [1].
In contrast, in Morocco, fve species of the Phlebotomus genus are involved in the transmission of leishmaniasis. Te frst one belonging to the Phlebotomus (P) subgenus is P. papatasi incriminated in the transmission of L. major; the second species belonging to the subgenus Paraphlebotmus is P. sergenti which is responsible for the transmission of L. tropica; the last three species belonging to the subgenus Larroussius are P. perniciosus, P. ariasi, and P. longicuspis. Tese three species are involved in the transmission of L. infantum, the causative agent of sporadic cutaneous forms and the severe visceral forms.
From a strategic point of view, a National Program for the Control of Leishmaniasis (NPCL) has been designed since 1997 to control the epidemiological situation of leishmaniasis in the country. Currently, its main goal is to eliminate all forms of leishmaniasis by 2030, based on a set of strategic axes: (a) free therapeutic management of cases; (b) control of the animal reservoir, particularly dogs and rodents; (c) vector control; (d) continuous training of health personnel; and (e) intersectoral collaboration and information, education, and communication [3,9].
Tus, the present study is part of the entomological surveillance and vector control of the NPCL. It aims to provide decision makers with basic data on the phlebotomine fauna circulating at the level of the province of El Hajeb in the center of Morocco where autochthonous cases of cutaneous and visceral leishmaniasis have been recorded.  (Figure 1).

Study
Te climate of El Hajeb is semihumid of the Mediterranean type; it is characterized by a hot and dry summer and a mild and rainy winter. Te average annual rainfall is about 572 mm and the average annual temperature is about 17°C with a maximum of 35°C in July and a minimum of 4°C in January [11].
Te choice of the trapping stations was based on epidemiological justifcations. Indeed, we based our work on retrospective studies of the epidemiological situation of leishmaniasis in this province during the last fve years [12]. Tese data allowed us to direct our survey eforts towards localities that have been afected by these vector-borne diseases.
Te support of local authorities and the collaboration of community agents and inhabitants allowed us to agree to trap in six stations (fve houses and one uncontrolled public landfll) in families with a history of leishmaniasis. Tese are the Sidi Mbarek station and the Aït Oufella station in the rural commune of Laqsir where autochthonous cases of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to the health authorities; the Aït Rbaa station in the rural commune of Bitit where cases of VL and CL have also been reported; the station of Aït Brahim where the surveys were carried out in the public landfll located near the locality where infantile VL cases were notifed; the station of Aïn Taoujdate where cases of CL were declared; and the station of Aït Naaman in the commune of El Hajeb where no case of leishmaniasis was reported was chosen as a control station.

Trapping Technique and Specimen Collection.
Te sampling of sand fies took place during the year 2019 in six stations in the province of El Hajeb in central Morocco. For the collection of sand fies, we used sticky paper traps (white A4 papers, soaked in castor oil on both sides) during the whole night [13]. During each capture session, 40 sheets were used and deposited in the afternoon at sunset and recovered the next morning at sunrise. Tese sticky traps were placed in the same location throughout the trapping period. Sand fies were captured in each locality on a bimonthly basis throughout the survey period. Te surveys were planned for one year from April 2019 to January 2020, April to January being the period of activity of sand fies in Morocco [14].
Te main biotopes that were surveyed were manure and animal burrows, chicken houses, stables, and near vegetation and rocks, as well as holes and inside and outside the house. Tus, we also equipped a mini mobile microscope adapted to our smartphone to photograph the sand fies captured in the feld.

Laboratory Work.
Te collected sand fies were transported to the laboratory where they were sorted, counted, sexed, and preserved in 70% ethanol solution. Te sand fies were thinned with a Marc-André solution and mounted between the slide and coverslip and then placed under the microscope for morphological identifcation using the sand fy identifcation keys published by the Ministry of Health [3], based on the pharynx and external genitalia in males and the spermatheca and cibarium in females. Tus, the identifcation of sand fies of the Larroussius subgenus was also carried out by referring to Leger and Depaquit [15] and Boussaa [16] and those of the subgenus Paraphlebotomus subgenus following Depaquit's method [17,18].

Data Analysis.
Based on the available sources, references, and records, the sand fy fauna has never been studied before in El Hajeb. Te characteristics of the captured fauna were determined by calculating six parameters, namely, (i) Abundance (A) which represents the total collected count of each species. (ii) Te relative abundance of each species (RA) is estimated by dividing the number collected of a given species by the total count of all species collected multiplied by 100: (iii) Species richness (S) represents the total number of species in the stand. (iv) Te diversity of the phlebotomine stand is expressed by the Shannon diversity index "H′" and the Evenns equitability index "E," where where Pi is the proportion of the total number of species samples where Hmax:LnS.
(v) Sex ratio (SR) is the ratio of males to females: (vi) Te density (D) is represented by the number of individuals per unit area of oil paper. It is expressed as the number of sand fies per square meter per night: where D: density; Ph: number of sand fies; and S: surface.

Results and Discussion
From an ecoepidemiological point of view, the province of El Hajeb constitutes a very interesting feld of study because of its geographical proximity to epidemic foci of cutaneous leishmaniasis in the provinces of Sefrou [19] and Moulay Yacoub [20]. In addition, recent studies have also shown the existence of autochthonous cases of CL and VL in the province of El Hajeb [12] whose responsible vectors are not yet recognized. Indeed, entomological data do not exist on the phlebotomine fauna circulating in this region. However, the identifcation of the species responsible for the transmission of leishmaniasis diseases in the afected localities is

Province of EI Hajeb
Communal division  Te Scientifc World Journal a fundamental step for the vector control activities and the prevention of these diseases, specifcally the severe forms that threaten humans and dogs [21]. It is in this perspective that the present work was carried out, exploring for the frst time the existing fauna and identifying the species incriminated in the transmission of this disease.

Diversity, Species Richness, and Sex Ratio of Sand Flies
Caught. A total of 14950 sand fies were captured, of which 78.3% (11715) were males and 21.7% (3235) were females. Te abundance, diversity, species richness, and sex ratio of the sand fy population captured at the six stations are presented in Table 1.
Across the region, twelve species were identifed with a diference between stations, and the sex ratio was 4.07 : 0.25. In fact, the ratio of males to females was in favor of males in the stations of Aït Taoujdate (2.8 : 0.36), Sidi Mbarek (4.04 : 0.25), Aït Oufella (8.23 : 0.12), Aït Rbaa (2.85 : 0.35), and Aït Naaman (1.95 : 0.52). In contrast, in the locality of Aït Brahim, females were collected more than males (0.73 : 1.35). In addition, the diversity of sand fies in El Hajeb was calculated using the Shannon diversity index "H′" and Evenns equitability index "E" (Table 1). Te fauna of the localities of Aït Rbaa and Sidi Mbarek had the greatest biodiversity (11 and 12 species out of 12), while that of Aït Brahim showed the least biodiversity (7 species out of 12).
Te analysis of the Shannon diversity index shows that the H′ values in the stations of Aït Naaman (H′ � 1.75) and Aït Brahim (H′ � 1.54) are closer to each other, while they are diferent in terms of maximum value of diversity. Tis means that the distribution of species at the level of the station of Aït Brahim in spite of containing fewer species (7 species) is better than that in the station of Aït Naaman (10 species).
Tus, the values of the diversity index in all the stations studied are a little diferent from each other with a diversifed population over the whole study area. Tis indicates the presence of a more stable ecosystem at El Hajeb. It should also be noted that the most diversifed station was that of Aït Naaman with a value of E � 0.76. Tis is due to an approximation between its diversity value H and its maximum theoretical value H′. Tis explains why the reproductive potential of the phlebotomine population seems to be the same, while the lowest diversity value was recorded in the station of Aït Oufella with a value of E � 0.265.

Inventory and Relative Abundance of the Identifed Sand
Flies Species. Of the 14590 sand fies collected, 14070 were identifed morphologically, and the rest of the sand fies (520) gravid females were retained for possible future molecular identifcation. Te detailed results of the identifcation of 14070 sand fies are presented in Table 2.
On the other hand, 24 species of sand fies have been described in Morocco, fve of which are known for their vectorial capacities to transmit the leishmaniasis which represents a public health problem [6,22]. Tese species are (a) P. papatasi, the vector of CL major which is widespread in the south and southeast of the country [3,23,24], (b) P. sergenti which is responsible for the transmission of cutaneous leishmaniasis to L. tropica for which most of the recorded cases are reported in the center of the kingdom [24,25], and (c) the other three species are P. perniciosus, P. ariasi, and P. longicuspis which have been proven as vectors of severe forms of the disease, especially VL for which most cases have been reported in the north [3,26].
In our study, 12 species were identifed, which represents 50% of the Moroccan sand fy species. Among these 12 species, fve were considered as vectors of the disease. Tey were also collected in the region of El Hajeb and represent 91.6% of the sand fies captured and are present in 83% of the sites surveyed (5/6). Tese include P. sergenti, P. papatasi, P. longicuspis, P. ariasi, and P. perniciosus.
On the other hand, the analysis of the results obtained showed that there is cohabitation between the 3 species: P. papatasi, P. sergenti, and P. longicuspis. Although the province of El Hajeb is located in the center of Morocco, this coexistence was also noted by Boussaa et al. [26] in their study conducted in the south and north of the country.
In another aspect, the analysis of the results in terms of abundance for each station revealed that the most important abundance was recorded at the station of Aït Oufella with 6833 specimens (46.8%), the station of Aïn Taoujdate with 3023 specimens (20.7%), and the station of Aït Rbaa with 2083 specimens (14.3%), then the station of Aït Naaman with 1044 (9.6%) and of Sidi M'barek with 1407 specimens (7.2%), and fnally the station of Aït Brahim with 200 specimens (1.4%). However, it was noticed that the abundance of fauna was very important compared to the low incidence of leishmaniasis recorded in the region [12]. Tis fnding has been found in other studies in other regions of Morocco [27][28][29][30]. Tis is mainly related to the ecology of the parasite rather than to the distribution of the vector [30]. Tus, recent studies have shown that the sand fy is called "Chniwla" by the local population and by health professionals in the region [7][8][9].

Seasonal Activity and Monthly Fluctuation of Fauna in El
Hajeb. Te results of the monthly activity of the three most abundant species in the six stations, Ain Taoujdate (AT), Sidi Mbarek (SM), Aït Oufella (AO), Aït Rbaa (AR), Aït Naaman (AN), and Aït Brahim (AB), are presented in Figure 2. Indeed, diferences have been noticed in the monthly evolution of each species.
At the Aïn Taoujdate station, species of the genus Phlebotomus, particularly of the subgenus Larroussius, were the most abundant. Although the peak of activity occurred in August and September, seasonal activity lasted for two      seasons, starting in April in the early spring and ending in October in the late summer, indicating that the life span of sand fies is relatively long, allowing sand fies to reproduce once or twice during this period. Indeed, the results show that the seasonal abundance of adults of P. longicuspis recorded a signifcant peak in activity during the summer months of July, August, and September. Similarly, the species P. perniciosus and P. sergenti showed a similar pattern, but with a single peak in August (Figure 2(a)). For the Sidi Mbarek station, P. longicuspis, P. sergenti, and P. papatasi were the most commonly encountered species. Te seasonal activity of adults of P. longicuspis refected three peaks: a frst peak in June and then the number of sand fies decreased steadily in July as the climate became warmer and then increased slightly to mark a second peak at the end of September and October, but the last peak was more important than the previous one in November, while the monthly abundance of P. sergenti and P. papatasi showed a bimodal peak pattern: one in July and the other in September which corresponds to the hot summer season that is favorable for sand fy reproduction (Figure 2(b)).
Concerning the station of Aït Oufella, the analysis of the monthly seasonal activity of the identifed sand fies showed that the most encountered species belonged to the genus Phlebotomus, in particular P. longicuspis, which showed a bimodal variation, the frst peak being in July and the second in September, whereas P. sergenti and P. perniciosus showed only one peak in September when the climate is warm (Figure 2(c)). It seems therefore that at this station, the summer season is the best breeding period for the diferent species of sand fies.
At the Aït Rbaa station, the sand fy activity ranged from April to November. Te seasonal abundance of adults of P. longicuspis peaked in May, and then, the number of sand fies increased slightly in July and September as the climate warmed up, reaching its peak in November. Adult P. sergenti activity followed a similar pattern, but peaked at the end of April, then in July, and fnally in November (Figure 2(d)). Tis means that the summer period ofers more opportunities for sand fies to breed since activity covers the months of July, August, and September, indicating that the life span of the sand fy is longer in this season compared to autumn when the sand fy activity is limited only to the month of November.
At the Aït Brahim station, the sand fy activity started in May and the adults of P. papatasi showed a single peak in July, while adults of P. bergeroti also showed a single peak but in August (Figure 2(f )).
In the majority of the locations prospected, sand fies started to appear in April and disappeared by the end of November. Tis could be explained by the moderate climate of the El Hajeb region which is of the Mediterranean type characterized by a hot summer ofering sand fies the possibility of breeding during the whole period of July-

Te Scientifc World Journal
August-September, whereas when the climate became colder in autumn, the life span of the sand fies is short with a single peak of activity in November.
On the whole, the analysis of the monthly and seasonal activities of the sand fies population of the entire region of El Hajeb shows that the maximum total density was recorded in the summer and autumn seasons, particularly in the months of July, August, September, and November, density which stood at, respectively, 31 ph/m 2 /night, 53 ph/ m 2 /night, 101 ph/m 2 /night, and 21 ph/m 2 /night (Figures 3  and 4).
Te monthly activity of the fve most abundant species incriminated in the transmission of leishmaniasis in El Hajeb province (P. longicuspis (79.4%), P. sergenti (7.2%), P. perniciosus (2.7%), P. papatasi (2.0%), and P. bergeroti (1.1%)) ranged from March to November. Te highest abundance was recorded during the months of July, August, September, and November. Te total catches of sand fies during the year show that there are two peaks: the frst and most important one was in summer when the climate is warm and the second one in autumn.
An analysis of monthly seasonal species-specifc activity ( Figure 4) shows that P. longicuspis ( Figure 5) was the most abundant in all stations, except the station of Ait Brahim. Tis species represents 79.7% of the sand fy population in the region of El Hajeb, and its activity extends from April to November with three peaks. Te frst peak begins in June, and the number of sand fies continues to increase to reach its maximum in September where it will mark its second peak which is the most important, and then, the number decreases steadily in October and increases slightly to record a third peak that was less important in November. Indeed, this species was found in all stations and during the whole study period but with very important abundance in the two localities of Aïn Taoujdate and Aït Oufella.
On the other hand, the long period of activity and high abundance of these confrmed VL vector species represent a threat and indicate the potential risk of VL transmission in the said province. Tese results corroborate the study of Al-Koleeby et al. [31] in which the most important peak is located in September, but diverge on the other hand in which this species can show a pattern with a single peak as is the case of the province of Chichaoua [32] or with a pattern of two peaks as is the case of the province of Zagora [31].
Similarly, this species showed a three-peak pattern in our study site. Tis can be explained by the ability of P. longicuspis to adapt to the environmental conditions of each region. In addition, 53% of these species were captured in the locality of Aït Oufella whose altitude is 581 m. In this context, Guernaoui et al. [29] showed that this species is very abundant between the altitudes of 600 m and 799 m.
Concerning P. perniciosus, it should frst be noted that the morphological identifcation of this species showed that almost all captured males are in typical form with a bifd apex penis ( Figure 6). Tis species was captured from April to November, and its seasonal trend refects a bimodal pattern with two peaks (Figure 4). Te frst peak was in July-August and the second peak was in November. In addition, this species was not found in the Aït Brahim station although it was collected during the entire study period in the other stations. In this context, studies conducted in the vicinity of El Hajeb have revealed that the seasonal evolution of this species takes the form of two peaks [19].
Te species P. sergenti (Figure 7) was encountered during the entire study period since this species is the only proven vector of L. tropica in Morocco. It was also caught in all stations and with very high abundance in the localities of Aït Oufella and Aït Rbaa. Its period of activity ranges from April to November with a biphasic pattern (Figure 4), the frst one in June-July and the other one in September. Our results confrm those found in the province of Fez [33] which revealed that the seasonal activity of this species is bimodal.
Te species P. Papatasi ( Figure 8) showed a monophasic pattern with a single peak in July (Figure 2). Tis vector species of L. major, especially in southern Morocco and which has long been considered adapted to the arid climate [24], is also adapted to the temperate climate that prevails in the center of the country since our results show the presence of this species from April to November and in the majority of stations (5/6), but with a high abundance in the stations of Aït Oufella and Aït Brahim. Tis result was also revealed by studies conducted in the provinces near El Hajeb [19] where it was also collected from April to November.
On the other hand, other species belonging to the genus Phlebotomus were encountered in El Hajeb but with weak abundance. Tis is the case in particular for P. ariasi, P. alexandri, and P. bergeroti. Te latter was caught with malformations for which the males were found to have a three-lobed paramere with a larger upper lobe and a lateral lobe with 3 spatulate terminal spines instead of 2 spines (Figure 9).
From another perspective, sand fies of the genus Sergentomyia represent 6.7% of the specimens collected, belonging to two subgenera: Sergentomyia and Grassomyia. Tese are S. minuta (Figure 10), S. schwetzi, S. fallax, S. antennata, and S. dreyfusi. Tese species prefer, according to Boussaa et al. [34], the altitudes between 800 m and 1000 m. Similarly, Guernaoui et al. [29] state that only the species S. minuta persists at the altitudes of 1200-2000 m. Tis was confrmed in our study particularly in the station of Aït Naaman which is located at an altitude of 1150 m and whose inventory of sand fies captured in this station revealed that the most abundant species belonged to the genus Sergentomyia, notably S. minuta (Figure 10), which represents 65.9%.
Te activity period of S. minuta ranges from April to November with a bimodal pattern in two peaks, the frst one which is the most important in August and the second one in September ( Figure 10). Tis species was most abundant in the locality of Aït Naaman where we collected 90.2% of the total number of these species.
Within this framework, it is very useful to point out that the selection of the station of Aït Naaman was carried out to compare the fauna of this station where no case of leishmaniasis was declared with the other stations afected by the said disease. In this framework, our results show that most of the specimens met in this station are of the Sergentomyia genus which explains the absence of leishmaniasis cases in this locality. Indeed, the species of the Sergentomyia genus are not yet proven to be involved in the transmission of leishmaniasis [1,4,35]. Nevertheless, the absence of leishmaniasis cases should not eliminate the potential risk of transmission since the vector species of L. infantum (P. longicuspis) and L. tropica (P. sergenti) have also been found. It appears that the inventory of phlebotomine fauna in El Hajeb was very important in terms of quantity and quality. Te majority of the species collected are proven to be involved in the transmission of the leishmaniasis disease, notably species belonging to the subgenus: (a) Larroussius, especially the species P. longicuspis, P. perniciosus, and

Conclusions
In summary, this study presents for the frst time the circulating sand fy fauna in the El Hajeb region of central Morocco. It provides basic data on the abundance, biodiversity, seasonality of sand fies, and their risk periods of disease transmission. It was shown that the species of sand fies involved in the transmission of leishmaniasis (P. papatasi, P. sergenti, P. longicuspis, and P. perniciosus) occupy the frst place in terms of predominance. Tese results serve as a very important tool for the decision makers of the National Leishmaniasis Control Program to focus their vector control actions towards the periods of high risk of transmission which coincide with the periods of peak density, especially in the summer and autumn seasons. Tus, entomological surveillance must be accompanied by awareness-raising and information activities for the inhabitants to avoid the health threats presented by sand fies and prevent the spread of leishmaniasis to other localities of El Hajeb in central Morocco.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest.